The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation

Abstract

Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer-disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrindisrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the "barbell shapes" of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure. (Blood. 2011;118(6): 1641-1652) IntroductionBlood platelets, like erythrocytes, must withstand high shear forces during circulation. Retaining their discoid shape is critical to platelets, because their small size and shape cause them to be propelled by blood flow to the endothelial surface, where they are positioned to readily sense and respond to vascular damage. To provide structural support and prevent gross deformations as they circulate, mature platelets contain a robust membrane skeleton that is formed by spectrin molecules, adducin, and actin filament barbed ends. [1][2][3] Two thousand spectrin tetramers, 200-nm-long head-tohead assemblies of ␣␤ heterodimers, compose the bulk of this 2D network. Although less is known about how the spectrin-actin network forms and connects to the plasma membrane in platelets relative to erythrocytes, certain differences between the 2 membrane skeletons have been recognized. First, spectrin strands comprising the platelet membrane skeleton interconnect on the ends of long actin filaments originating from the cytoplasm instead of short actin oligomers. [3][4][5] Therefore, the platelet spectrin lattice and its associated actin filaments assemble into a continuous ultrastructure. Second, tropomodulins do not appear to have a major role in capping actin filament pointed ends, as occurs in erythrocytes. 6,7 Instead, a substantial number of these ends exist free or are capped by Arp2/3 in the res...

Essential role of class II PI3K-C2α in platelet membrane morphology

“…27 It has recently been shown that the cortical actin-myosin-spectrin system brings external compressive forces during barbell-shaped proplatelet formation and determines platelet size. 15,19,33 This is important in the context of nonmuscle myosin MYH9-related diseases affecting platelets, 33 and possibly Bernard-Soulier syndrome, and may explain the large size and decreased number of platelets of these patients. Interestingly, membrane skeleton proteins such as b-spectrin and filamin, as well as myosin IIA and moesin, were identified in a screen of proteins able to interact with PI3P.…”

“…The spectrin-based membrane skeleton is critical for biophysical membrane properties, both in platelets and megakaryocytes, 27,28 and is essential for the conversion from preplatelets to barbell-shaped proplatelets and to mature platelets. 15,19 The membrane skeleton of WT and PI3K-C2a WT/D1268A platelets was isolated, and its composition analyzed by immunoblotting. Although the level of cortical polymerized actin was normal, a significant decrease of cortical spectrin and myosin was observed in PI3K-C2a WT/D1268A platelets compared with in WT platelets ( Figure 5B).…”

“…Platelet shaping and sizing resulting from reversible conversion of round-shaped platelets into barbell-shaped proplatelets also occurs in the bloodstream, where they undergo successive rounds of fission via membrane and cytoskeleton remodeling to produce mature platelets. [13][14][15][16][17][18][19][20] To gain insight into the physiological role of PI3K-C2a in platelet production and function, we used a mouse line heterozygous for a germ-line kinase-inactive knock-in (KI) mutation (D1268A) in the endogenous Pik3c2a gene. These mice with a heterozygous inactivation of PI3K-C2a were viable and fertile but showed aberrant platelet formation with critical defect in platelet membrane morphology and dynamics and an enrichment of barbell-shaped proplatelets in the bloodstream.…”

Microtubule and cortical forces determine platelet size during vascular platelet production

“…5h). We have also recently shown that barbell proPLTs treated with the spectrin disrupting peptide spα2N1 are unable to retain their barbell shape and collapse into a circular prePLT form 14 . These observations confirm that elongating microtubule bundles in the peripheral coil are being deformed by the external compressive pressure of the actin-myosin-spectrin cortex during proPLT conversion.…”

UROS and ALAS-2: erstwhile partners in crime

“…In this issue of Blood, To-Figueras and colleagues present convincing genetic and biochemical evidence that the clinical phenotype of the rare disorder is markedly affected by the coinheritance of an activating mutation in the erythroid-specific isoform of ␦-aminolevulinic acid synthase. 1 M ost physicians look on the porphyrias with a jaundiced eye. These disorders are rare, the clinical phenotypes are complex and highly variable, the laboratory evaluation is expensive and difficult to access, and the results are usually negative or inconclusive.…”